Alzheimer disease (AD) is the most common form of progressive dementia in adults over the age of 60 years. Multiple missense mutations have been reported in the presenilin-1 (PS1) and presenilin-2 (PS2) proteins that are linked to familial AD (FAD). Presenilins are known to function as the catalytic subunit of the Q-secretase complex and FAD mutations in presenilins affect A(5 amyloid precursor protein (APP) processing, leading to the accumulation of Ap42 peptide and amyloid plaque formation in AD brains. In addition to abnormal APP processing, several FAD mutations in presenilins have been linked to abnormal calcium (Ca2+) signaling. Our laboratory recently discovered that presenilin holoproteins function as [unreadable] endoplasmic reticulum (ER) Ca2+ leak channels and that FAD mutations in presenilins affected this function. FAD PS1 mutation M146V, L166P, A246E, E273A, G384A, P436Q and PS2-N1411 abolished ER Ca2+ leak while PAD PS1-A79V and D257A (gamma secretase catalytic mutant)had no effect. PS1-AE9, however, enhanced channel activity (see appendix). Our findings potentially provided an explanation for Ca2+ signaling abnormalities resulting from FAD mutations in presenilins. The goal of my thesis project is to establish a connection between presenilins FAD mutations and ER Ca2+ signaling. These data will help to evaluate the "Ca2+ hypothesis of AD" and will contribute to selecting optimal strategies for treatment of AD. Specifically, I propose: [unreadable] 1. To evaluate the function of ER Ca2+ leak pathway in lymphoblast from human FAD patients. [unreadable] Samples of human lymphoblast from FAD patients with mutations in presenilins, APP and tau will be obtained from the National Cell Repository for Alzheimer's Disease (NCRAD). The human lymphoblast from healthy individuals will also be obtained and used as controls. The function of ER Ca2+ leak pathway in these cells will be evaluated in Ca2+ imaging experiments. [unreadable] 2. To analyze the effects of NCRAD FAD mutations in presenilin-1 on ER Ca2+ leak function. [unreadable] In these experiments I will focus on the analysis of 8 additional FAD PS1 missense mutants (M139V,K239E, V261F, P264L, R269G, C410Y, A426P and A431E). Selected FAD mutants will be expressed in Sf9 cells by baculovirus infection and their Ca2+ channel activity will be analyzed in planar lipid bilayers. The FAD mutants will be transfected into PS1/2 double-knockout (DKO) mouse embryonic fibroblasts (MEF) and their ability to rescue impaired ER Ca2+ leak pathway will be evaluated in Ca2+ imaging experiments. [unreadable] [unreadable] [unreadable] [unreadable]